CN103114042B - Dothideomycete sp. mutant strain and method for catalytic production of betulin alcohol by using same - Google Patents
Dothideomycete sp. mutant strain and method for catalytic production of betulin alcohol by using same Download PDFInfo
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- CN103114042B CN103114042B CN201210560282.1A CN201210560282A CN103114042B CN 103114042 B CN103114042 B CN 103114042B CN 201210560282 A CN201210560282 A CN 201210560282A CN 103114042 B CN103114042 B CN 103114042B
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Abstract
The invention discloses a dothideomycete sp. mutant strain and a method for catalytic production of betulin alcohol by using same. The mutant strain is dothideomycete sp. UV 14 and is preserved in the China Center for Type Culture Collection, and the preservation Number is CCTCC. No. M2012523. The method for producing betulin alcohol comprises the steps that dothideomycete sp. is activated, then inoculated into a potato dextrose liquid medium containing sodium chloride and cultured for 1-3 days; the dothideomycete sp. is subject to reaction for 12-60 hours in the medium after betulin and dimethyl sulfoxide are added inside, and then is filtered, and the filtrate is extracted by using ethyl acetate; thalli obtained by filtering is freeze-dried and then milled and extracted three times by adding ethyl acetate, organic phases obtained are combined and the solvent is removed under vacuum and coarse betulin alcohol product is subject to column chromatography to obtain the product betulin alcohol. The dothideomycete sp. mutant strain and the method for catalyze-producing betulin alcohol by using same have the advantages of high regioselectivity, mildness in reaction conditions, high conversion efficiency, short reaction time, high target product yield and the like, and is environment-friendly.
Description
Technical field
The invention belongs to natural product and applying biological catalytic field, be specifically related to the method that birch keto-alcohol is prepared in a kind of capsule mould mutant strain and the oxidation of catalysis betulin regioselectivity thereof.
Background technology
Betulin is a kind of feather fan alkanes pentacyclic triterpene compound, there is anti-inflammatory and antiviral isoreactivity, and demonstrate the mechanism of action different from other medicines, tumour cell is also had to certain toxicity (chemistry of forest product and industry, 2009,29 (1), 87), but lower biological activity and selectivity etc. have hindered its application clinically.For this reason, taking betulin as lead compound, it is carried out to structural modification and become study hotspot (Natural Product Reports, 2006,23,394) in recent years to improve its biological activity.Birch keto-alcohol is the carbonyl derivative after betulin 3-hydroxyl oxidize.Quantity research shows greatly, and the biological activity of birch keto-alcohol is far above betulin.For example, birch keto-alcohol to the toxicity of lung cancer NSCLC-N6 cell far above betulin (Phytochemistry, 2004,65,1159); The 3-hydroxyl of betulin, after oxidative modification, has obviously strengthened its inducing mouse melanoma cells differentiation activity (Journal of Natural Products, 2002,65,645).Birch keto-alcohol is also the crucial building block (Molecules, 2011,16,2443) that preparation has the isomery birch keto-alcohol (allobetulone) of significant application value simultaneously.
Although many plants are as all contained birch keto-alcohol (tropical and subtropical plant journal, 2007,15 (3), 249 in large handle Chinese ilex (Ilex macropoda) and mangrove plant scyphiphora hydrophyllacea (Scyphiphora hydrophyllacea) etc.; Archives of Pharmacal Research, 2002,25,617), but content is very low, has greatly limited research and the application of this compound.Therefore, must develop the easy new technology of preparing birch keto-alcohol and novel process.Betulin is extract before the biosynthesizing of birch keto-alcohol, and in plant, content is higher, especially in Japanese birch bark its content up to 25%.Therefore taking betulin cheap, that be easy to get as raw material, preparing birch keto-alcohol by chemical/biological catalysis method is a synthetic route that has application potential.
In betulin molecule, there are three active function groups (3-hydroxyl, 28-hydroxyl and C
20-C
29two keys), because the selectivity of chemical method is poor, therefore as want to 3-hydroxyl wherein carry out selective oxidation modify time, conventionally need loaded down with trivial details protection and deprotection steps, and conventionally need to use stoichiometric, the disagreeableness oxygenant CrO of environment
3and KMnO
4deng.Although utilize biocatalysis and bio-transformation means can effectively avoid the above-mentioned shortcoming of chemical method, 28-hydroxyl is primary hydroxyl, and 3-hydroxyl is secondary hydroxyl, and the former is sterically hindered relatively little, therefore the former is conventionally active than the latter, is more easily modified.In fact the biological catalyst of, having reported at present also can only catalysis 28-hydroxyl oxidize and hydroxylating (ProcessBiochemistry 2011, the 46:1-15 in upper other sites of ring; Enzyme and Microbial Technology, 2009,45:175-180; Journal of Applied Microbiology, 2011,110:90-97), still cannot realize the regioselectivity oxidation of SA 3-hydroxyl.Recently, we screen the seat capsule mould HQ 316564 of the synthetic birch keto-alcohol of a strain energy regioselectivity catalysis betulin 3-hydroxyl oxidize, and under optimum condition, productive rate is 43%(Journal of Molecular Catalysis B:Enzymatic, 2013,88:32-35).But taking this original strain as biological catalyst, transformation efficiency is lower, the reaction times long (need preculture bacterial strain 3 days, add substrate then react 6 days), and productive rate is not high.
Summary of the invention
The problem existing for prior art, utilize ultraviolet accidental mutating technology to carry out mutagenesis to original strain seat capsule mould Dothideomycete sp.HQ 316564, screening obtains the mutant strain of the synthetic birch keto-alcohol of a strain efficient catalytic catalysis betulin regioselectivity oxidation.The object of the present invention is to provide the method for this capsule mould mutant strain and the synthetic birch keto-alcohol of catalysis betulin regioselectivity oxidation thereof.
Object of the present invention is achieved through the following technical solutions:
A kind of seat capsule mould mutant strain, this mutant strain was seat capsule mould (Dothideomycete sp.) UV 14, on December 11st, 2012, be preserved in Chinese Typical Representative culture collection center, be called for short CCTCC, preserving number: CCTCC NO.M 2012523, preservation address: Wuhan, Wuhan University.The colonial morphology of this mutant strain is as shown in Fig. 2 (b).
The method of utilizing above-mentioned seat capsule mould catalytic production birch keto-alcohol, comprises the steps:
(1) a seat capsule mould (Dothideomycete sp.) is after UV 14 is activated, and in physiological saline, configuration concentration is 1 × 10
7the seed liquor of individual spore/mL, is seeded in the potato liquid of glucose substratum of sodium chloride-containing, cultivates 1 ~ 3 day;
(2) add after substrate betulin and solubility promoter dimethyl sulfoxide (DMSO), regulate pH to 5.0 ~ 9.0 of substratum, under 23 ~ 38 DEG C, 160r/min, react after 12 ~ 60 hours, filter, filtrate extracts three times (equal-volume refers to filtrate volume and equates) by isopyknic ethyl acetate; Filter gained thalline after freeze-drying, add the ethyl acetate of 1/2 filtrate volume mill and extract three times, merge gained organic phase, under vacuum, except desolventizing, after column chromatography, obtain product birch keto-alcohol.
Preferably, the activation of the described seat of step (1) capsule mould (Dothideomycete sp.) UV 14 is on the potato glucose agar medium of the pH 7.0 of sodium chloride-containing, under 28 ° of C, activates 6 days.Preferably, the formula of the potato glucose agar medium of described sodium chloride-containing is: 2% glucose, 20% potato, 1.5% agar and 0.3% sodium-chlor.
Preferably, the inoculum size of the described seed liquor of step (1) is 1%.
Preferably, the formula of the potato liquid of glucose substratum of the described sodium chloride-containing of step (1) is: 2% glucose, 20% potato and 0.3% sodium-chlor.
Preferably, the described culture condition of step (1) is 5.0 ~ 7.0,23 ~ 38 DEG C of initial pH, 140 ~ 180r/min.
Preferably, the concentration of the described dimethyl sulfoxide (DMSO) of step (2) is 0.2 ~ 1.0%(v/v).
Preferably, the concentration of the described betulin of step (2) is 100mg/L.
The present invention compared with prior art, has advantages of as follows:
1) utilize seat capsule mould Dothideomycete sp.UV14 cell as sterically hindered higher, active lower 3-position secondary hydroxy oxidation in the high regioselectivity ground catalysis of catalyzer energy betulin.
2) the synthetic birch keto-alcohol using seat capsule mould Dothideomycete sp.UV14 cell as catalyzer, compared with original strain Dothideomycete sp., has that biocatalysis efficiency is high, the reaction times is short, object product yield advantages of higher.
3) the inventive method regioselectivity is high, technique is simple, has avoided protection and the deprotection steps of much time power, and has reaction conditions gentleness, eco-friendly advantage.
Brief description of the drawings
Fig. 1 is the reaction formula that betulin is converted into birch keto-alcohol.
Fig. 2 is original bacterium Dothideomycete sp.HQ 316564(a) and mutant strain (Dothideomycetesp.) UV 14(b) in the potato glucose agar medium of 3% sodium-chlor, pH 7.0, under 28 ° of C, cultivate the colonial morphology after 3 days.
Fig. 3 is the typical liquid chromatographic figure that analyzes of betulin and birch keto-alcohol (retention time of betulin and birch keto-alcohol be respectively 10.09 and 11.98min).
Embodiment
Further illustrate the present invention by embodiment, but be not limited to embodiment.
Embodiment 1
Seat capsule mould (Dothideomycete sp.) UV 14 is at potato glucose agar medium (2% glucose of sodium chloride-containing, pH 7.0,20% potato, 1.5% agar, 0.3% sodium-chlor) on, under 28 ° of C, activate after 6 days, in physiological saline, configure seed liquor (1 × 10
7individual spore/mL), be seeded to by 1% inoculum size in the potato liquid of glucose substratum (2% glucose, 20% potato, 0.3% sodium-chlor) of 50mL sodium chloride-containing, pH 6.0, under 28 ° of C, 160r/min, cultivate 3 days; Add subsequently 5mg betulin and 0.4%(v/v) dimethyl sulfoxide (DMSO), regulate medium pH to 6.0, under 33 ° of C, 160r/min, react; After 48 hours, filter, filtrate extracts three times by isopyknic ethyl acetate, filter gained thalline after freeze-drying, add the ethyl acetate of 1/2 filtrate volume mill and extract three times, merge gained organic phase, under vacuum, except desolventizing, obtain product birch keto-alcohol crude product; Add subsequently the dissolve with methanol of 0.5mL, utilize after centrifugal liquid chromatography (color atlas as shown in Figure 3) to measure production concentration, calculating productive rate according to product calibration curve is 44%.
Embodiment 2
Seat capsule mould Dothideomycete sp.UV 14 (2% glucose on the potato glucose agar medium of sodium chloride-containing, pH 7.0,20% potato, 1.5% agar, 0.3% sodium-chlor), under 28 ° of C, activate after 6 days, in physiological saline, configure seed liquor (1 × 10
7individual spore/mL), be seeded to by 1% inoculum size in the potato liquid of glucose substratum (2% glucose, 20% potato, 0.3% sodium-chlor) of 50mL sodium chloride-containing, pH 6.5, under 28 ° of C, 160r/min, cultivate 1.5 days; Add subsequently 5mg betulin and 0.6%(v/v) dimethyl sulfoxide (DMSO), regulate medium pH to 6.5, under 33 ° of C, 160r/min, react; After 48 hours, filter, filtrate, with isopyknic ethyl acetate extraction three times, is filtered gained thalline after freeze-drying, adds 1/2 filtrate volume ethyl acetate mill and extract three times, merges gained organic phase, under vacuum, except desolventizing, obtains product birch keto-alcohol crude product; Add subsequently the dissolve with methanol of 0.5mL, utilize liquid chromatogram measuring production concentration after centrifugal, calculating productive rate according to product calibration curve is 49%.
Seat capsule mould Dothideomycete sp.UV 14 is at potato glucose agar medium (2% glucose of sodium chloride-containing, pH 7.0,20% potato, 1.5% agar, 0.3% sodium-chlor) on, under 28 ° of C, activate after 6 days, in physiological saline, configure seed liquor (1 × 10
7individual spore/mL), be seeded to by 1% inoculum size in the potato liquid of glucose substratum (2% glucose, 20% potato, 0.3% sodium-chlor) of 50mL sodium chloride-containing, pH 5.5, under 28 ° of C, 160r/min, cultivate 1.5 days; Add subsequently 5mg betulin and 0.2%(v/v) dimethyl sulfoxide (DMSO), regulate medium pH to 6.5, under 33 ° of C, 160r/min, react; After 48 hours, filter, filtrate, with isopyknic ethyl acetate extraction three times, is filtered gained thalline after freeze-drying, adds 1/2 filtrate volume ethyl acetate mill and extract three times, merges gained organic phase, under vacuum, except desolventizing, obtains product birch keto-alcohol crude product; Add subsequently the dissolve with methanol of 0.5mL, utilize liquid chromatogram measuring production concentration after centrifugal, calculating productive rate according to product calibration curve is 57%.
Embodiment 4
Seat capsule mould Dothideomycete sp.UV 14 is at potato glucose agar medium (2% glucose of sodium chloride-containing, pH 7.0,20% potato, 1.5% agar, 0.3% sodium-chlor) on, under 28 ° of C, activate after 6 days, in physiological saline, configure seed liquor (1 × 10
7individual spore/mL), be seeded to by 1% inoculum size in the potato liquid of glucose substratum (2% glucose, 20% potato, 0.3% sodium-chlor) of 50mL sodium chloride-containing, pH 5.5, under 23 ° of C, 160r/min, cultivate 1.5 days; Add subsequently 5mg betulin and 0.2%(v/v) dimethyl sulfoxide (DMSO), regulate medium pH to 7.0, under 33 ° of C, 160r/min, react; After 48 hours, filter, filtrate, with isopyknic ethyl acetate extraction three times, is filtered gained thalline after freeze-drying, adds 1/2 filtrate volume ethyl acetate mill and extract three times, merges gained organic phase, under vacuum, except desolventizing, obtains product birch keto-alcohol crude product; Add subsequently the dissolve with methanol of 0.5mL, utilize liquid chromatogram measuring production concentration after centrifugal, calculating productive rate according to product calibration curve is 54%
Embodiment 5
Seat capsule mould Dothideomycete sp.UV 14 is at potato glucose agar medium (2% glucose of sodium chloride-containing, pH 7.0,20% potato, 1.5% agar, 0.3% sodium-chlor) on, under 28 ° of C, activate after 6 days, in physiological saline, configure seed liquor (1 × 10
7individual spore/mL), be seeded to by 1% inoculum size in the potato liquid of glucose substratum (2% glucose, 20% potato, 0.3% sodium-chlor) of 50mL sodium chloride-containing, pH 5.5, under 28 ° of C, 160r/min, cultivate 1.5 days; Add subsequently 5mg betulin and 0.4%(v/v) dimethyl sulfoxide (DMSO), regulate medium pH to 7.0, under 28 ° of C, 160r/min, react; After 48 hours, filter, filtrate, with isopyknic ethyl acetate extraction three times, is filtered gained thalline after freeze-drying, adds 1/2 filtrate volume ethyl acetate mill and extract three times, merges gained organic phase, under vacuum, except desolventizing, obtains product birch keto-alcohol crude product; Add subsequently the dissolve with methanol of 0.5mL, utilize liquid chromatogram measuring production concentration after centrifugal, calculating productive rate according to product calibration curve is 63%.
Embodiment 6
Seat capsule mould Dothideomycete sp.UV 14 is at potato glucose agar medium (2% glucose of sodium chloride-containing, pH 7.0,20% potato, 1.5% agar, 0.3% sodium-chlor) on, under 28 ° of C, activate after 6 days, in physiological saline, configure seed liquor (1 × 10
7individual spore/mL), be seeded to by 1% inoculum size in the potato liquid of glucose substratum (2% glucose, 20% potato, 0.3% sodium-chlor) of 50mL sodium chloride-containing, pH 5.5, under 28 ° of C, 160r/min, cultivate 1.5 days; Add subsequently 5mg betulin and 0.4%(v/v) dimethyl sulfoxide (DMSO), regulate medium pH to 7.0, under 33 ° of C, 160r/min, react; After 36 hours, filter, filtrate, with isopyknic ethyl acetate extraction three times, is filtered gained thalline after freeze-drying, adds 1/2 filtrate volume ethyl acetate mill and extract three times, merges gained organic phase, under vacuum, except desolventizing, obtains product birch keto-alcohol crude product; Add subsequently the dissolve with methanol of 0.5mL, utilize liquid chromatogram measuring production concentration after centrifugal, calculating productive rate according to product calibration curve is 62%.
Embodiment 7
Seat capsule mould Dothideomycete sp.UV 14 is at potato glucose agar medium (2% glucose of sodium chloride-containing, pH 7.0,20% potato, 1.5% agar, 0.3% sodium-chlor) on, under 28 ° of C, activate after 6 days, in physiological saline, configure seed liquor (1 × 10
7individual spore/mL), be seeded to by 1% inoculum size in the potato liquid of glucose substratum (2% glucose, 20% potato, 0.3% sodium-chlor) of 50mL sodium chloride-containing, pH 5.5, under 28 ° of C, 160r/min, cultivate 1.5 days; Add subsequently 5mg betulin and 0.4%(v/v) dimethyl sulfoxide (DMSO), regulate medium pH to 7.0, under 33 ° of C, 160r/min, react; After 48 hours, filter, filtrate, with isopyknic ethyl acetate extraction three times, is filtered gained thalline after freeze-drying, adds 1/2 filtrate volume ethyl acetate mill and extract three times, merges gained organic phase, under vacuum, except desolventizing, obtains product birch keto-alcohol crude product; Add subsequently the dissolve with methanol of 0.5mL, utilize liquid chromatogram measuring production concentration after centrifugal, calculating productive rate according to product calibration curve is 72%.
Claims (9)
1. a seat capsule mould mutant strain, is characterized in that, this mutant strain is seat capsule mould (Dothideomycete sp.) UV14, on December 11st, 2012, be preserved in Chinese Typical Representative culture collection center, be called for short CCTCC, preserving number: CCTCC NO.M2012523.
2. the method for utilizing seat capsule mould mutant strain catalytic production birch keto-alcohol described in claim 1, is characterized in that, comprises the steps:
(1) seat capsule mould CCTCC NO.M2012523 activated after, in physiological saline, configuration concentration is 1 × 10
7the seed liquor of individual spore/mL, is seeded in the potato liquid of glucose substratum that contains sodium-chlor, cultivates 1～3 day;
(2) add after substrate betulin and solubility promoter dimethyl sulfoxide (DMSO), regulate pH to 5.0～9.0 of substratum, under 23～38 DEG C, 160r/min, react after 12～60 hours, filter, filtrate extracts three times by isopyknic ethyl acetate; Filter gained thalline after freeze-drying, add the ethyl acetate of 1/2 filtrate volume mill and extract three times, merge gained organic phase, under vacuum, except desolventizing, after column chromatography, obtain product birch keto-alcohol.
3. method according to claim 2, is characterized in that, the activation of the described seat of step (1) capsule mould CCTCCNO.M2012523 is on the potato glucose agar medium of sodium chloride-containing, pH7.0, activates 6 days at 28 DEG C.
4. method according to claim 3, is characterized in that, the formula of the potato glucose agar medium of the described sodium chloride-containing of step (1) is: 2% glucose, 20% potato, 1.5% agar and 0.3% sodium-chlor.
5. according to the method described in claim 2 or 3 or 4, it is characterized in that, the inoculum size of the described seed liquor of step (1) is 1%.
6. according to the method described in claim 2 or 3 or 4, it is characterized in that, the formula of the potato liquid of glucose substratum of the described sodium chloride-containing of step (1) is: 2% glucose, 20% potato and 0.3% sodium-chlor.
7. according to the method described in claim 2 or 3 or 4, it is characterized in that, the described culture condition of step (1) is initial pH5.0～7.0,23～38 DEG C, 140～180r/min.
8. according to the method described in claim 2 or 3 or 4, it is characterized in that, the concentration of the described dimethyl sulfoxide (DMSO) of step (2) is 0.2～1.0%v/v.
9. according to the method described in claim 2 or 3 or 4, it is characterized in that, the concentration of the described betulin of step (2) is 100mg/L.
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| CN101084234A (en) * | 2004-09-10 | 2007-12-05 | 康乃尔研究基金会有限公司 | Betulinol derivatives as anti-cancer agents |
| WO2008070347A2 (en) * | 2006-10-27 | 2008-06-12 | Regents Of The University Of Minnesota | Betulin-peptide conjugates |
| CN102634560B (en) * | 2012-04-12 | 2013-10-30 | 华南理工大学 | Method for preparing Birch ketol through Dothideales mycete region-selectivity catalysis of betulin |
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